The impact of Mmu17 non-Hsa21 orthologous genes in the Ts65Dn mouse model of Down syndrome: the “gold standard” refuted

Background: Despite successful preclinical treatment studies to improve neurocognition in the Ts65Dn mouse model of Down syndrome (DS), translation to humans has failed. This raises questions about the appropriateness of the Ts65Dn mouse as the “gold standard” for DS research. Here we used the novel Ts66Yah mouse that carries both an extra chromosome and the identical segmental Mmu16 trisomy as Ts65Dn without the Mmu17 non-orthologous region. Methods: Forebrains from embryonic day 18.5 Ts66Yah and Ts65Dn mice, along with euploid littermate controls, were used for gene expression and pathway analyses. Behavioral experiments were performed in neonatal and adult mice. Since male Ts66Yah mice are fertile, parent of origin transmission of the extra chromosome was studied. Results: We mapped 45 protein coding genes to the Ts65Dn Mmu17 non-orthologous, among which 71-82 % are expressed during forebrain development. Several of these genes were uniquely overexpressed in Ts65Dn embryonic forebrain; this produced major differences in dysregulated genes and pathways. Despite these genome-wide differences, the primary Mmu16 trisomic effects were highly conserved in both models, resulting in several commonly dysregulated disomic genes and pathways. Delays in motor development, communication and olfactory spatial memory were present in Ts66Yah but more pronounced in Ts65Dn neonates. Adult Ts66Yah mice showed working memory deficits and sex-specific effects in exploratory behavior and spatial hippocampal memory, while long-term memory was preserved. These deficits were milder when compared to Ts65Dn mice. Conclusions: Our findings suggest that triplication of the non-orthologous Mmu17 genes significantly contributes to the phenotype of the Ts65Dn mouse and may explain why preclinical trials that used this model have unsuccessfully translated to human therapies. Total RNA was isolated from the developing E18.5 forebrain using the NuceloMag® RNA/DNA high throughput extraction kit following the manufacturer’s instructions (Macherey-Nagel, Bethlehem, PA). RNA was processed and hybridized on the Clariom S high throughput arrays. Eighty nine arrays were used (18 EupTs65Dn, 23 Ts65Dn, 24 EupTs66Yah and 25 Ts66Yah) and each array corresponded to labeled cDNA from one sample. Balanced numbers of male and female embryos were used in these studies. Analyses were performed using unpaired t-tests. A Benjamini-Hochberg false discovery rate (FDR) of 10% was used for multiple comparison correction of differently expressed (DEX) genes. The marginally expressed genes (MEX) (expression ratios <0.8 and >1.2 and raw p-values <0.01) were used for pathway analyses. Pathway analysis was performed using Ingenuity Pathway Analysis (IPA) database and the Database for Annotation, Visualization, and Integrated Discovery (DAVID).